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Substrate recognition by the AAA+ chaperone ClpB

Abstract

The AAA+ protein ClpB cooperates with the DnaK chaperone system to solubilize and refold proteins from an aggregated state. The substrate-binding site of ClpB and the mechanism of ClpB-dependent protein disaggregation are largely unknown. Here we identified a substrate-binding site of ClpB that is located at the central pore of the first AAA domain. The conserved Tyr251 residue that lines the central pore contributes to substrate binding and its crucial role was confirmed by mutational analysis and direct crosslinking to substrates. Because the positioning of an aromatic residue at the central pore is conserved in many AAA+ proteins, a central substrate-binding site involving this residue may be a common feature of this protein family. The location of the identified binding site also suggests a possible translocation mechanism as an integral part of the ClpB-dependent disaggregation reaction.

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Figure 1: Identification and characterization of ClpB-interacting peptides.
Figure 2: Preloading of ClpB-B1/2A with peptide B1 prevents further substrate association.
Figure 3: Peptides bind to the first AAA domain of ClpB.
Figure 4: Identification of a potential substrate-binding site.
Figure 5: ClpB pore mutants exhibit reduced chaperone activities and their ATPase activities are stimulated less by peptides and substrates.
Figure 6: Reduced binding of ClpB pore mutants to peptides and substrates in vitro.
Figure 7: The substrate interactions of ClpB pore mutants are affected in vivo.
Figure 8: Direct interaction between the ClpB pore site and substrates revealed by crosslinking.

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References

  1. Ogura, T. & Wilkinson, A.J. AAA+ superfamily ATPases: common structure—diverse function. Genes Cells 6, 575–597 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Vale, R.D. AAA proteins. Lords of the ring. J. Cell Biol. 150, F13–F19 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Neuwald, A.F., Aravind, L., Spouge, J.L. & Koonin, E.V. AAA+: a class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 9, 27–43 (1999).

    CAS  PubMed  Google Scholar 

  4. Guo, F., Maurizi, M.R., Esser, L. & Xia, D. Crystal structure of ClpA, an Hsp100 chaperone and regulator of ClpAP protease. J. Biol. Chem. 277, 46743–46752 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Bochtler, M. et al. The structures of HsIU and the ATP-dependent protease HsIU-HsIV. Nature 403, 800–805 (2000).

    Article  CAS  PubMed  Google Scholar 

  6. Li, J. & Sha, B. Crystal structure of E. coli Hsp100 ClpB nucleotide-binding domain 1 (NBD1) and mechanistic studies on ClpB ATPase activity. J. Mol. Biol. 318, 1127–1137 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Yu, R.C., Hanson, P.I., Jahn, R. & Brunger, A.T. Structure of the ATP-dependent oligomerization domain of N-ethylmaleimide sensitive factor complexed with ATP. Nat. Struct. Biol. 5, 803–811 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Lenzen, C.U., Steinmann, D., Whiteheart, S.W. & Weis, W.I. Crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein. Cell 94, 525–536 (1998).

    Article  CAS  PubMed  Google Scholar 

  9. Zhang, X. et al. Structure of the AAA ATPase p97. Mol. Cell 6, 1473–1484 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. Dougan, D.A., Mogk, A., Zeth, K., Turgay, K. & Bukau, B. AAA+ proteins and substrate recognition, it all depends on their partner in crime. FEBS Lett. 529, 6–10 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. Song, H.K. et al. Mutational studies on HslU and its docking mode with HslV. Proc. Natl. Acad. Sci. USA 97, 14103–14108 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zeth, K. et al. Structural analysis of the adaptor protein ClpS in complex with the N-terminal domain of ClpA. Nat. Struct. Biol. 9, 906–911 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Leonhard, K., Stiegler, A., Neupert, W. & Langer, T. Chaperone-like activity of the AAA domain of the yeast Yme1 AAA protease. Nature 398, 348–351 (1999).

    Article  CAS  PubMed  Google Scholar 

  14. Smith, C.K., Baker, T.A. & Sauer, R.T. Lon and Clp family proteases and chaperones share homologous substrate-recognition domains. Proc. Natl. Acad. Sci. USA 96, 6678–6682 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Goloubinoff, P., Mogk, A., Peres Ben Zvi, A., Tomoyasu, T. & Bukau, B. Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network. Proc. Natl. Acad. Sci. USA 96, 13732–13737 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Glover, J.R. & Lindquist, S. Hsp104, Hsp70 and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94, 73–82 (1998).

    Article  CAS  PubMed  Google Scholar 

  17. Zolkiewski, M. ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation. A novel multi-chaperone system from Escherichia coli. J. Biol. Chem. 274, 28083–28086 (1999).

    Article  CAS  PubMed  Google Scholar 

  18. Mogk, A. et al. Identification of thermolabile E. coli proteins: prevention and reversion of aggregation by DnaK and ClpB. EMBO J. 18, 6934–6949 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Motohashi, K., Watanabe, Y., Yohda, M. & Yoshida, M. Heat-inactivated proteins are rescued by the DnaK. J-GrpE set and ClpB chaperones. Proc. Natl. Acad. Sci. USA 96, 7184–7189 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Konieczny, I. & Liberek, K. Cooperative action of Escherichia coli ClpB protein and DnaK chaperone in the activation of a replication initiation protein. J. Biol. Chem. 277, 18483–18488 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Thomas, J.G. & Baneyx, F. Roles of the Escherichia coli small heat shock proteins IbpA and IbpB in thermal stress management: comparison with ClpA, ClpB, and HtpG in vivo. J. Bacteriol. 180, 5165–5172 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Sanchez, Y., Taulien, J., Borkovich, K.A. & Lindquist, S. Hsp104 is required for tolerance to many forms of stress. EMBO J. 11, 2357–2364 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hong, S.W. & Vierling, E. Mutants of Arabidopsis thaliana defective in the acquisition of tolerance to high temperature stress. Proc. Natl. Acad. Sci. USA 97, 4392–4397 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Schirmer, E.C., Glover, J.R., Singer, M.A. & Lindquist, S. HSP100/Clp proteins: a common mechanism explains diverse functions. Trends Biochem. Sci. 21, 289–296 (1996).

    Article  CAS  PubMed  Google Scholar 

  25. Weibezahn, J., Schlieker, C., Bukau, B. & Mogk, A. Characterization of a trap mutant of the AAA+ chaperone ClpB. J. Biol. Chem. 278, 32608–32617 (2003).

    Article  CAS  PubMed  Google Scholar 

  26. Rüdiger, S., Germeroth, L., Schneider-Mergener, J. & Bukau, B. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 16, 1501–1507 (1997).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Woo, K.M., Kim, K.I., Goldberg, A.L., Ha, D.B. & Chung, C.H. The heat-shock protein ClpB in Escherichia coli is a protein-activated ATPase. J. Biol. Chem. 267, 20429–20434 (1992).

    CAS  PubMed  Google Scholar 

  28. Cantor, C.R. & Schimmel, P.R. Biophysical Chemistry: Part III (W.H. Freeman, San Francisco, 1135–1139, 1980).

    Google Scholar 

  29. Chin, J.W., Martin, A.B., King, D.S., Wang, L. & Schultz, P.G. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc. Natl. Acad. Sci. USA 99, 11020–11024 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Hattendorf, D.A. & Lindquist, S.L. Cooperative kinetics of both Hsp104 ATPase domains and interdomain communication revealed by AAA sensor-1 mutants. EMBO J. 21, 12–21 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Schlee, S., Groemping, Y., Herde, P., Seidel, R. & Reinstein, J. The chaperone function of ClpB from Thermus thermophilus depends on allosteric interactions of its two ATP-binding sites. J. Mol. Biol. 306, 889–899 (2001).

    Article  CAS  PubMed  Google Scholar 

  32. Mogk, A. et al. Roles of individual domains and conserved motifs of the AAA+ chaperone ClpB in oligomerization, ATP-hydrolysis and chaperone activity. J. Biol. Chem. 278, 15–24 (2003).

    Google Scholar 

  33. Lee, S. et al. The Structure of ClpB. A molecular chaperone that rescues proteins from an aggregated state. Cell 115, 229–240 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Wang, J. et al. Crystal structures of the HslVU peptidase–ATPase complex reveal an ATP-dependent proteolysis mechanism. Structure 9, 177–184 (2001).

    Article  CAS  PubMed  Google Scholar 

  35. Sousa, M.C. et al. Crystal and solution structures of an HslUV protease-chaperone complex. Cell 103, 633–643 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Niwa, H., Tsuchiya, D., Makyio, H., Yoshida, M. & Morikawa, K. Hexameric ring structure of the ATPase domain of the membrane-integrated metalloprotease FtsH from Thermus thermophilus HB8. Structure 10, 1415–1423 (2002).

    Article  CAS  PubMed  Google Scholar 

  37. Krzywda, S. et al. The crystal structure of the AAA domain of the ATP-dependent protease FtsH of Escherichia coli at 1.5 Å resolution. Structure 10, 1073–1083 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Chaney, M. et al. Binding of transcriptional activators to σ 54 in the presence of the transition state analog ADP-aluminium fluoride: insights into activator mechanochemical action. Genes Dev. 15, 2282–2294 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lee, S.Y. et al. Regulation of the transcriptional activator NtrC1: structural studies of the regulatory and AAA+ ATPase domains. Genes Dev. 17, 2552–2563 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Yamada-Inagawa, T., Okuno, T., Karata, K., Yamanaka, K. & Ogura, T. Conserved pore residues in the AAA protease, FtsH, are important for proteolysis and its coupling to ATP hydrolysis. J. Biol. Chem. 278, 50182–50187 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Squires, C.L., Pedersen, S., Ross, B.M. & Squires, C. ClpB is the Escherichia coli heat shock protein F84. 1. J. Bacteriol. 173, 4254–4262 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Giese, K.C. & Vierling, E. Changes in oligomerization are essential for the chaperone activity of a small heat shock protein in vivo and in vitro. J. Biol. Chem. 277, 46310–46318 (2002).

    Article  CAS  PubMed  Google Scholar 

  43. Schlothauer, T., Mogk, A., Dougan, D.A., Bukau, B. & Turgay, K. MecA, an adaptor protein necessary for ClpC chaperone activity. Proc. Natl. Acad. Sci. USA 100, 2306–2311 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Arsène, F. et al. Role of region C in regulation of the heat shock gene-specific sigma factor of Escherichia coli, σ32. J. Bacteriol. 181, 3552–3561 (1999).

    PubMed  PubMed Central  Google Scholar 

  45. McCarty, J.S. et al. Regulatory region C of the E. coli heat shock transcription factor, σ32, constitutes a DnaK binding site and is conserved among eubacteria. J. Mol. Biol. 256, 829–837 (1996).

    Article  CAS  PubMed  Google Scholar 

  46. Kramer, A. & Schneider-Mergener, J. Synthesis and screening of peptide libraries on continuous cellulose membrane supports. Methods Mol. Biol. 87, 25–39 (1998).

    CAS  PubMed  Google Scholar 

  47. Mogk, A. et al. Refolding of substrates bound to small Hsps relies on a disaggregation reaction mediated most efficiently by ClpB/DnaK. J. Biol. Chem. 278, 31033–31042 (2003).

    Article  CAS  PubMed  Google Scholar 

  48. Laufen, T. et al. Mechanism of regulation of Hsp70 chaperones by DnaJ co-chaperones. Proc. Natl. Acad. Sci. USA 96, 5452–5457 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Tomoyasu, T., Mogk, A., Langen, H., Goloubinoff, P. & Bukau, B. Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol. Mol. Microbiol. 40, 397–413 (2001).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank D. Dougan, K. Turgay and E. Weber-Ban for discussions and critical reading of the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Leibnizprogramm and Bu617/14-1) and the Fond der Chemischen Industrie to B.B. and A.M.

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Correspondence to Bernd Bukau or Axel Mogk.

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Supplementary information

Supplementary Fig. 1

The peptide-induced oligomerization of ClpB-B1/2A is ATP-dependent. (PDF 250 kb)

Supplementary Fig. 2

ClpB pore mutants exhibit no oligomerization and structural defects. (PDF 1034 kb)

Supplementary Fig. 3

ClpB interacts with a short 14-mer oligopeptide. (PDF 14 kb)

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Schlieker, C., Weibezahn, J., Patzelt, H. et al. Substrate recognition by the AAA+ chaperone ClpB. Nat Struct Mol Biol 11, 607–615 (2004). https://doi.org/10.1038/nsmb787

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